Appearance and regression of rat pouch tissue

Fibrosis, a consequence of tissue repair, can become a final common pathway to organ failure, if progressive. Prevention and regression of organ fibro sis represent targets of considerable interest. The natural fate of fibrosi s differs among various tissues being either persistent, progressive or reg ressive. Cellular and molecular responses involving myofibroblasts (myoFb), a phenotypically transformed fibroblast-like cell of considerable function al diversity, is involved in collagen turnover at sites of repair, where th ey govern the fate of fibrosis. Insights gained from the natural regression of established fibrous tissue may offer strategies to remove unwanted fibr osis in failing organs, In the present study, we addressed the temporal sequence to various compone nts of collagen synthesis and degradation involved in the appearance and su bsequent regression of pouch tissue induced in the rat by subcutaneous inje ction of air followed by instillation of the phorbol ester croton oil. Pouc h tissue was collected on day 2, 4, 10, 14, 21, 28 and 35 (n = 6 at each ti me point). Activities of matrix metalloproteinase-l (MMP-1) and tissue inhi bitor of MMP-1 (TIMP-1) were determined by zymography and reverse zymograph y, respectively: collagen accumulation by hydroxyproline concentration: gen e expression of TIMP-1 or tissue inhibitor of MMP-1, type I collagen and tr ansforming growth factor-beta(1) (TGF-beta(1)) by in situ hybridization; TG F-beta(1) concentration by sandwich enzyme-linked immunosorbant as say (ELI SA ); and myoFb and its phenotypes by immunohistochemistry using antibodies to alpha-smooth muscle actin (a-Sh;Wi, vimentin or desmin. During pouch ti ssue formation, we found: ii) pouch weight increased progressively from day 2 to day 14 and then declined progressively thereafter; (2) type I collage n mRNA expression, barely detectable at day 2, increased at day 4, together with tissue hydroxyproline concentration (P<0.05) reaching a peak on day 1 0, and gradually decreased thereafter in association with declining tissue hydroxyproline concentration; (3) mRNA expression and concentration of TGF- beta(1), detectable at day 2, significantly (P<0.05) increased at day 4, re ached a peak at day 10, and gradually declined thereafter; (4) MMP-1 activi ty, low at day 2, increased continually over the course of 35 days; (5) TIM P-1 mRNA, detectable at day 2 and significantly (P<0.05) increased at day 4 , gradually decreased thereafter: (6) activity of TIMP-1 increased continuo usly from day 2 to day 14 and then was markedly reduced thereafter; and (7) myoFb were first observed in pouch tissue at day 4 and became more extensi ve thereafter with their phenotype changing over time, Early appearing myoF b (day 4, 10, 14, and 21) expressed alpha-SMA and vimentin (VA phenotype), while later appearing cells (day 28 and 35) additionally expressed desmin ( VAD phenotype). Thus, in croton oil-induced rat pouch model, the subcutaneo us accumulation of pouch tissue hydroxyproline over the course of 10 days i s initially associated with a VA-positive myoFb phenotype and its transcrip tion of TGF-P,, type I collagen and TIMP-1. Beyond day 10, a regression of pouch tissue collagen begins in association with the appearance of a VAD-po sitire myoFb phenotype and progressive increase in MMP-1 activity as the ex pression of TIMP-1 and TGF-P, are withdrawn. Regression of established fibr osis in failing organs may, therefore, be attainable through manipulation o f myoFb phenotype and/or enhanced collagen degradation relative to collagen synthesis. (C) 1999 Academic Press.